CN102260653B - Preparation and application method of PEG recombinant pig-human urate oxidase fusion protein - Google Patents
Preparation and application method of PEG recombinant pig-human urate oxidase fusion protein Download PDFInfo
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Abstract
The invention provides a preparation method of PEG recombinant pig-human urate oxidase fusion protein, a method for preparing injecta and lyophilized injection by PEG recombinant pig-human urate oxidase fusion protein, and application for reducing content of blood uric acid. The invention further provides a nucleotide sequence of pig-human urate oxidase, which comprises vectors of nucleotide sequence and host cells containing the vectors. The PEG recombinant pig-human urate oxidase fusion protein provided with the invention has no immunogenicity substantially, maintains activity of urate oxidase before PEG and has good treatment effect for reducing the content of blood uric acid.
Description
Technical field
The invention belongs to field of biological pharmacy, be specifically related to a kind of preparation and application method thereof of PEGization restructuring pig-human urate oxidase fusion protein.
Background technology
UriKoxidase claims again urico-oxidase (Uricase, Urate Oxidase, EC 1.7.3.3, UO), can the oxidation of catalysis uric acid generate wallantoin, CO
2And H
2O
2All find to have the existence of this enzyme in the tissues such as the liver of most vertebrate, kidney, but the mankind and some primates are because transgenation loses the synthetic ability [1] that active urico-oxidase is arranged.
Uric acid is the catabolic final product of purine in the human body, by renal excretion, excretes with urine.Uric acid mainly exists with sodium-salt form in vivo, and extremely sodium salt solubleness in water is very low for the uric acid of free form.If purine metabolic disturbance produces excessive uric acid in the body, or uric acid excretion is obstructed, and uric acid in blood concentration raise, thereby cause the diseases such as hyperuricemia and gout.Malignant hematologic disease and chemotherapy of tumors also can cause blood uric acid concentration to raise.
Hyperuricemia and gout can not only cause arthroncus, also can bring out and increase the weight of disorders of lipid metabolism, diabetes, increase cardiovascular and cerebrovascular diseases, metabolism disturbance syndrome and kidney disease patient's mortality ratio.In the last few years, along with the variation of China's people's lives mode and dietary structure, goat was suffered from the patient and was increased sharply, and presented the trend of rejuvenation.Morbidity approaches developed country's levels such as American-European up to about 4%, has become common disease, the frequently-occurring disease of serious threat China people ' s health.
At present, for the treatment of goat, normal operation promotes uric acid excretion or suppresses the synthetic medicine of uric acid.Uricosuric has probenecid class medicine, by suppressing uriniferous tubules uric acid is heavily absorbed, and increases its drainage.Suppressing the synthetic main medicine of uric acid is Zyloric.It is the competitive inhibitor of XOD, can suppress xanthine oxidase and change into uric acid, reduces so that uric acid is synthetic.But it can cause the precursor xanthine concentration of uric acid to raise.And xanthic water-soluble lower than uric acid, xanthine injury of the kidney and deposition can occur in some patient.Zyloric and probenecid class medicine have obvious liver renal toxicity, and curative effect all not ideal enough [2].
Urico-oxidase is the invalid goat patient's for the treatment of routine treatment ideal medicament.Have the lethal mutation of 2 places in the human body in the urate oxidase gene, they lay respectively at the 33rd and the 187th amino acids residue of urico-oxidase aminoacid sequence, become termination codon (CGA → TGA) by arginine residues.In order to obtain a kind of human urico-oxidase preparation that uses that is suitable for, the present invention use one section more similar to Human genome and have the gene of the pig urico-oxidase of better vigor to replace nonfunctional part in the Human genome, prepare the pig-human urate oxidase mosaic gene, use this genes produce preparation to can be used for clinical pig-human urate oxidase fusion protein.
Clinical observation shows that if the protein medicaments multiple dosing, its drug effect often is difficult to keep.Its reason is that protein itself is exactly natural antigen, can stimulate body to produce antibody after being expelled in the body, affect the result for the treatment of in later stage by antigen antibody reaction, also can cause allergic reaction in the time of seriously.One of effective way that addresses this problem is exactly protein to be carried out Pegylation (PEGization) modify, and to reach the reduction immunogenicity, prolongs the purpose of circulating half-life.PEG is a kind of uncharged linear hydrophilic molecules, and it can form with the nonessential group covalent attachment of protein the barrier on the space conformation, avoids the antigenic determinant of foreign protein to induce generation antibody.Because mostly the substrate of enzyme is small-molecule substance, can pass the PEG barrier and be combined with reactive site, so PEGization can significantly not reduce the activity of enzyme.The present invention relates to a kind of method, can shield to greatest extent the antigenicity of pig-human urate oxidase, and can well preserve its enzyme activity, very high biological safety is also arranged.
Summary of the invention
The preparation method who the purpose of this invention is to provide PEGization restructuring pig-human urate oxidase fusion protein, with the method for preparing injection liquid and lyophilized injectable powder with PEGization restructuring pig-human urate oxidase, and this injection liquid or the application of powder injection in reducing Uric Acid Content.
The present invention also provides the nucleotide sequence of restructuring pig-human urate oxidase, comprises the carrier of this nucleotide sequence, and comprises the host cell of this carrier.
Technical scheme of the present invention:
A kind of restructuring pig-human urate oxidase of PEGization is characterized in that this binding substances comprises the covalently bound thing of restructuring pig-human urate oxidase and PEG, and the two is covalently bound by the amino-formate bond form; Described restructuring pig-human urate oxidase can be the tetramer; Covalently bound 8-12 PEG chain on each restructuring pig-human urate oxidase subunit, comparative optimization is 9-11 PEG chain, more preferably 9-10 PEG chain; Described PEG can be linear or branch shape, and the molecular weight of PEG can be 2-50KD, and comparative optimization is 5-20KD, more preferably 10KD; The nucleotide sequence of described restructuring pig-human urate oxidase can be the sequence in the sequence table 1, and its aminoacid sequence can be the sequence in the sequence table 2.
A kind of composition that reduces Plasma Uric Acid content is characterized in that comprising binding substances and the pharmaceutically acceptable carrier of described restructuring pig-human urate oxidase and PEG.The route of administration of said composition comprises intramuscular injection, intravenous injection, the red and swollen position of gout or position, uratoma place local injection etc.; Said composition can exist with dosage forms such as injection liquid or freeze-dried powders.
Described PEGization restructuring pig-human urate oxidase and the pharmaceutical composition that comprises this binding substances can be used in for the preparation of the medicine that reduces Plasma Uric Acid content, can be used for the clinically treatment of hyperuricemia and whole body and the topical therapeutic of goat.
A kind of nucleic acid molecule of the restructuring pig-human urate oxidase albumen of encoding, its nucleotide sequence can be the sequence in the sequence table 1; A kind of host cell that contains the plasmid vector of the described restructuring pig-human urate oxidase albumen of encoding and contain this carrier; This carrier can be plasmid vector, and host cell can be intestinal bacteria.
The below is described in detail the present invention:
Restructuring pig-human urate oxidase fusion protein provided by the invention, especially PEG with the restructuring pig-human urate oxidase binding substances, essentially no immunogenicity, and substantially keep urico-oxidase active, preferably can keep 85% enzymic activity, comparative optimization keep 90% enzymic activity, preferredly keep 95%, even be the enzymic activity more than 98%.
Restructuring pig-human urate oxidase provided by the invention is fusion rotein, and 1-222 amino-acid residue is by the sequence encoding of pig urate oxidase gene from the N end, and 223-305 aminoacid sequence encoded by people's urate oxidase gene.The amino acid full length sequence of this fusion rotein is seen sequence table 2.The N end is preferably the X-222 nucleotide sequence of pig nucleotide sequence N end, and wherein X is 1-10, and the X of comparative optimization is 2-9, and further preferred X is 3-8, and further preferred X is 4-7, and most preferred X is 7.In the used pig urate oxidase gene of the present invention sequence, there are 3 positions variant with external report pig urico-oxidase sequence.They are respectively that the 111bp place becomes C by T, and the 456bp place becomes T by C, and the 669bp place becomes G by T.Pig urate oxidase gene sequence of the present invention is the sequence of subordinate list 3.But the aminoacid sequence of its coding does not change.223-305 aminoacid sequence of fusion rotein encoded by people's urate oxidase gene.People's urico-oxidase full length sequence sees attached list 4.The coding gene sequence of the 223-305 amino-acid residue of preferred groups of people's urico-oxidase nucleotide sequence behaviour urico-oxidase nucleotide sequence.In order to make things convenient for genetic manipulation, add that at 5 ' end of restructuring pig and human urate oxidase gene ACC makes it to downcut gene fragment with restriction endonuclease Nco I; Add the sequence ctc gag of restriction endonuclease Xho I at 3 ' end.
Pig and human restructuring urate oxidase gene can adopt the method for full gene synthetic, or adopts the method for RT-PCR to clone from pig and people's hepatic tissue respectively.If people's fresh liver tissue is difficult for obtaining the also N terminal sequence of the urate oxidase gene of the method clone pig of available RT-PCR, and obtain the C terminal sequence of people's urate oxidase gene with the method for synthetic.N terminal sequence and C terminal sequence are connected into restructuring pig-human urate oxidase mosaic gene.Recombination sequence is inserted in the T carrier, transform bacillus coli DH 5 alpha.Obtain recombination from the cloned plasmids carrier and be inserted into the pET28a plasmid vector, be built into expression plasmid, and transform e. coli bl21, obtain expressing engineering bacteria E.coli BL21/pET28a-PHUO.
Produce the expression engineering bacteria with the method for fermentation culture, treat that adding inductor alpha-lactose to working concentration when the OD value reaches 1.4 left and right sides is 5-6mmol/L.Inducing culture 5 hours.Be crushed to 98% above bacterial cell disruption with ultrasonic disruption or with high pressure homogenizer after receiving bacterium.Use respectively ion exchange column (DEAE Sepharose Fast Flow), drainage column (Phoenyl Sepharose 6 Fast Flow), gel-filtration column (Sephacryl S 300HR) carries out the expression product purifying.
The described restructuring pig-human urate oxidase of the application can be the tetramer.PEG chain with active group in the free amine group of the Lys side chain on tetramer surface and the mPEG-SC molecule carries out linked reaction.A covalently bound average out to 8-12 PEG chain on each restructuring pig-human urate oxidase subunit of the present invention, comparative optimization be 9-11 PEG chain, be more preferably 9-10 PEG chain; Described PEG can be linear or branch shape, and the molecular weight of PEG can be 2-50KD, and comparative optimization is 5-20KD, more preferably 10KD.The subunit of each pig-human urate oxidase fusion protein contains 28 free lysine residues, adds that end amino acid has 29 free amine groups.Modifying following its enzymic activity of 12 lysine residues does not obviously reduce.
The present invention also comprises a kind of composition of uric acid reducing content, it is characterized in that comprising above-described PEGization restructuring pig-human urate oxidase and pharmaceutically acceptable carrier.Described pharmaceutically acceptable carrier comprises the thinner of pharmaceutical field routine, vehicle etc.Non-intestinal drug delivery agent comprises injection etc., also comprises freeze-dried powder.Comparative optimization formulation of the present invention is injection, the freeze-dried powder that most preferably is, and wherein freeze-dried powder can redissolve by water for injection or specific solution, can keep the activity of former binding substances; Preferred route of administration comprises intramuscular injection, the red and swollen position of intravenous injection and gout local injection etc.Above-mentioned various formulation all can be according to the ordinary method preparation of pharmaceutical field.
According to results of animal, binding substances of the present invention is adapted at Uric Acid Content and is higher than normal value 50% and uses when above, and the dosage general control is in 2-8 unit/kg body weight.After being lower than normal value, Uric Acid Content stops using.The pitch time of secondary administration is more than 7 days.Each treatment cycle administration 2-3 time.
The invention provides a kind of nucleic acid molecule of the described restructuring pig-human urate oxidase albumen of encoding, its nucleotide sequence can be the sequence in the sequence table 1.
A kind of dna molecular carrier that contains the described restructuring pig-human urate oxidase albumen of encoding comprises cloning vector and expression vector, and preferred vector is plasmid vector, and wherein preferred cloning vector is pMD 18-T, and preferred expression vector is pET28a.
The invention provides a kind of host cell that comprises these carriers, comprise bacterium, fungi, vegetable cell, zooblasts etc. are preferably intestinal bacteria.
The invention provides the preparation method of the nucleic acid molecule of expressing the restructuring pig-human urate oxidase, described method comprises according to nucleotide sequence carries out full gene synthetic scheme, and RT-PCR amplification scheme and RT-PCR amplification pig urate oxidase gene add three kinds of schemes of synthetic people urate oxidase gene Partial Fragment.
The invention provides the method for the nucleic acid molecule of amplification restructuring pig-human urate oxidase, described method comprises the pcr amplification method.Can adopt conventional pcr amplification method, comprise the sequences Design synthetic primer according to this nucleic acid molecule.In the presence of an amount of target nucleic acid molecule and excessive Nucleotide and polysaccharase, add an amount of primer, by with repeatedly sex change of target nucleic acid molecule, with primer annealing and with a plurality of circulations of primer extension to subchain, thereby the target nucleic acid molecule index is increased.Product after the amplification is reclaimed by electrophoresis, be connected on the carrier, import in the host cell again.Amplified production is connected on the plasmid vector, according to the method for transformation of routine this plasmid vector is imported among bacillus coli DH 5 alpha or the BL21.Select the transformant of the nucleotide sequence that comprises the pig-human urate oxidase of recombinating, cryogenic freezing is preserved.
The invention provides the preparation method of restructuring pig-human urate oxidase fusion protein.Described method comprises that the carrier with the nucleotide sequence that contains this albumen transforms or transfection host cell, according to the characteristic of host cell and expression vector, selects suitable abduction delivering condition, and induced gene efficiently expresses this albumen.Described method comprises this albumen of method extraction purifying that adopts conventional extraction purifying protein in this area.The preferred chromatographic technique purifying protein that adopts.The invention provides the intermediate processing of unique restructuring pig-human urate oxidase.
The invention provides the method for preparing PEGization restructuring pig-human urate oxidase binding substances.With activated PEG product (mPEG-SC, the molecular weight: 10K) modify of purification of Recombinant pig-human urate oxidase with Beijing Kaizheng Biotech Engineering Development Co., Ltd.'s production.Pig-human urate oxidase to be reorganized be connected with PEG finish after with the method for gel permeation chromatography method or ultrafiltration, PEGization restructuring pig-human urate oxidase is separated with other small-molecule substance.
The invention provides the preparation method of PEGization restructuring pig-human urate oxidase injection and lyophilized injectable powder.With the preparation method of the restructuring of the PEGization behind purifying pig-human urate oxidase with protein medicaments preparation conventional on the pharmacology, be prepared into injection and lyophilized injectable powder.
Description of drawings
Figure A purifying protein SDS-PAGE figure
Figure A:1,2,3,4, swimming lane are that 1. 2. 3. 4. 4 stopple coupons of purifying protein distinguish called afters, and the protein content of loading is 10 μ g; The 5th swimming lane is albumen Marker; 6th, 7,8 swimming lanes are 3. number stopple coupon purifying protein, and applied sample amount is respectively 10 μ g, 20 μ g, 30 μ g.
Figure B purifying pig-human urate oxidase high performance liquid chromatography purity detecting
Specific embodiment
Below provide specific embodiment to set forth the present invention, but should not be construed to be the restriction the present invention.
Scheme one full gene synthetic schemes
The full gene that carries out the pig-human urate oxidase mosaic gene by following sequence is synthetic:
The sequence essentially consist is that the gene order of 1-222 amino-acid residue of N end is pig urate oxidase gene sequence.Hold 2-6 amino acid whose gene order to delete N, hold in 5 ' of gene order to add that ACC makes it to downcut gene fragment and recovery with restriction endonuclease Nco I.The coding gene sequence of the 223-305 amino-acid residue of sequence is people's urate oxidase gene sequence, and end adds the sequence ctc gag of restriction endonuclease Xho I, so that genetic manipulation.
Nucleotide sequence is:
1 ACC ATG GAC TAC AAA AAG AAT GAT GAG GTA GAG TTT
49 GTC CGA ACT GGC TAT GGG AAG GAT ATG ATA AAA GTT CTC CAT ATT CAG
97 CGA GAT GGA AAA TAT CAC AGC ATT AAA GAG GTG GCA ACT TCA GTG CAA
145 CTG ACT TTG AGC TCC AAA AAA GAT TAC CTG CAT GGA GAC AAT TCA GAT
193 GTC ATC CCT ACA GAC ACC ATC AAG AAC ACA GTT AAT GTC CTG GCG AAG
241 TTC AAC GGC ATC AAA AGC ATA GAA ACT TTT GCT GTG ACT ATC TGT GAG
289 CAT TTC CTT TCT TCC TTC AAG CAT GTC ATC AGA GCT CAA GTC TAT GTG
337 GAA GAA GTT CCT TGG AAG CGT TTT GAA AAG AAT GGA GTT AAG CAT GTC
385 CAT GCA TTT ATT TAT ACT CCT ACT GGA ACG CAC TTC TGT GAG GTT GAA
433 CAG ATA AGG AAT GGA CCT CCA GTT ATT CAT TCT GGA ATC AAA GAC CTA
481 AAA GTC TTG AAA ACA ACC CAG TCT GGC TTT GAA GGA TTC ATC AAG GAC
529 CAG TTC ACC ACC CTC CCT GAG GTG AAG GAC CGG TGC TTT GCC ACC CAA
577 GTG TAC TGC AAA TGG CGC TAC CAC CAG GGC AGA GAT GTG GAC TTT GAG
625 GCC ACC TGG GAC ACT GTT AGG AGC ATT GTC CTG CAG AAA TTT GCT GGG
673 CCC TAT GAC AAA GGT GAA TAC TTG ACC TCT GTG CAG AAG ACC CTC TGT
721 GAT ATC CAG GTG CTC TCC CTG AGC CGA GTT CCT GCG ATA GAA GAT ATG
769 GAA ATC AGC CTG CCA AAC ATT CAC TAC TTC AAC ATA GAC ATG TCC AAA
817 ATG GGT CTG ATC AAC AAG GAA GAG GTA TTG CTG CCA TTA GAC AAT CCA
865 TAT GGA AAA ATT ACT GGT ACA GTC AAG AGG AAG TTG TCT TCA AGA CTG
913 TGA CTC GAG
2, the PCR primer is synthetic
22-mer，UPPER PRIMER：
5’ACC ATG GAC TAC AAA AAG AAT G 3’
5 ' end adds restriction endonuclease Nco I restriction enzyme site.
22-mer，LOWER PRIMER：
5’CTC GAG TCA CAG TCT TGA AGA C 3’
5 ' end adds restriction endonuclease Xho I restriction enzyme site.
3, pcr amplification pig-human urate oxidase mosaic gene
4, amplified production is connected on the pMD18-T carrier, extracts plasmid DNA.
The scheme two RT-PCR scheme that increases
1, get pig liver tissue and carry out total RNA extraction, synthetic primer, sequence is:
Upstream primer:
5’ACC ATG GAC TAC AAA AAG AAT G 3’
Downstream primer:
5’AAA TTT CTG CAG GAC AAT GCT CC 3’
Take total RNA as template, use the N terminal sequence of the method amplification pig urate oxidase gene of RT-PCR.Be connected on the T carrier.Transform bacillus coli DH 5 alpha, extract plasmid DNA, reclaim goal gene with restriction endonuclease Nco I+Dra I.
2, in the process of people's operation on liver, obtain a little human liver tissue, carry out total RNA and extract, synthetic primer, sequence is:
Upstream primer:
5’AAA TTT GCT GGG CCC TAT GAC AAA GG 3’
Downstream primer:
5’CTC GAG TCA CAG TCT TGA AGA CAA C 3’
Take total RNA as template, use the C terminal sequence of the method amplification people urate oxidase gene of RT-PCR.Be connected on the T carrier.Transform bacillus coli DH 5 alpha, extract plasmid DNA, reclaim goal gene with restriction endonuclease Dra I+Xho I.
Hold dna fragmentation to be connected dna fragmentation to connect with ligase enzyme with C N.Then connect product with following primer amplification.
Upstream primer:
5’ACC ATG GAC TAC AAA AAG AAT G 3’
Downstream primer:
5’CTC GAG TCA CAG TCT TGA AGA CAA C 3’
Amplified production is connected on the T carrier.
Scheme three RT-PCR amplification+genes are synthetic
1, get pig liver tissue and carry out total RNA extraction, synthetic primer, sequence is:
Upstream primer:
5’ACC ATG GAC TAC AAA AAG AAT G 3’
Downstream primer:
5’AAA TTT CTG CAG GAC AAT GCT CC 3’
Take total RNA as template, use the N terminal sequence of the method amplification pig urate oxidase gene of RT-PCR.Be connected on the T carrier.Transform bacillus coli DH 5 alpha, extract plasmid DNA, reclaim goal gene with restriction endonuclease Nco I+Dra I.
2, carry out the synthetic of people's urate oxidase gene C terminal sequence by following sequence.
AAA TTT GCT GGG CCC TAT GAC AAA GGT GAA TAC TTG ACC TCT GTG CAG
AAG ACC CTC TGT GAT ATC CAG GTG CTC TCC CTG AGC CGA GTT CCT GCG
ATA GAA GAT ATG GAA ATC AGC CTG CCA AAC ATT CAC TAC TTC AAC ATA
GAC ATG TCC AAA ATG GGT CTG ATC AAC AAG GAA GAG GTA TTG CTG CCA
TTA GAC AAT CCA TAT GGA AAA ATT ACT GGT ACA GTC AAG AGG AAG TTG
TCT TCA AGA CTG TGA CTC GAG
Pcr amplification people urate oxidase gene C terminal sequence.Primer sequence is as follows:
Upstream primer:
5’AAA TTT GCT GGG CCC TAT GAC AAA GG 3’
Downstream primer:
5’CTC GAG TCA CAG TCT TGA AGA CAA C 3’
Be connected on the T carrier.Transform bacillus coli DH 5 alpha, extract plasmid DNA, reclaim goal gene with restriction endonuclease Dra I+Xho I.
Hold dna fragmentation to be connected dna fragmentation to connect with ligase enzyme with C N.Then connect product with following primer amplification.
Upstream primer:
5’ACC ATG GAC TAC AAA AAG AAT G 3’
Downstream primer:
5’CTC GAG TCA CAG TCT TGA AGA CAA C 3’
Amplified production is connected on the T carrier.
Select expression vector plasmid pET28a, expression host cell is Escherichia coli BL21.Building process is as follows:
1) double digestion of pET28a and pMD PHUO (pig-human urate oxidase) reaches enzyme and cuts the product electrophoresis, and reclaims fragment:
Nco I/Xho I double digestion pET28a, Nco I/Xho I double digestion pMD PHUO, the double digestion product carries out gel electrophoresis.Recovery is with the pET28a fragment at Nco I/Xho I point of contact and with the restructuring urico-oxidase mosaic gene fragment at Nco I/Xho I point of contact.
2) with the connection of the pET28a fragment at Nco I/Xho I point of contact and restructuring urico-oxidase mosaic gene fragment.
3) conversion of connection product: transform e. coli bl21 with connecting product.Transformed bacteria called after: Escherichia coli BL21/pET28a-PHUO (pig-human urate oxidase).
4) by dna sequencing, select the host cell that contains restructuring pig-human urate oxidase mosaic gene, freezing preservation, and transfer to the center preservation of Chinese Typical Representative culture collection.
The abduction delivering of embodiment 3, restructuring pig-human urate oxidase albumen
1. shaking flask is cultivated in a small amount
The single bacterium colony of picking e. coli bl21/pET28a-PHUO bacterium is inoculated in the LB+Kan nutrient solution of 10ml overnight incubation.Get saturated bacterium liquid 1ml in the 250mlLB+Kan nutrient solution, 37 ℃ of shaking culture 2.5h are to OD
600=1.4～1.5, add an amount of inductor alpha-lactose.Cultivate again 5h for 37 ℃, centrifugal receipts bacterium.Add-on to the inductor lactose is repeatedly optimized, and lactose concn is 1-10mmol/L when determining shaker fermentation, and preferred lactose concn is 5-6mmol/L.The 1L nutrient solution can obtain wet bacterium about 5 grams, and the wet bacterium of every gram obtains the thick enzyme of 400U.The protein electrophoresis result shows that target protein accounts for more than 30% of thalline soluble protein total amount.
2.150L fermentor tank amplification culture
On the basis of cultivating in a small amount, the parameters such as medium nutrient content, fermentation are optimized.The LB prescription of the preferred substratum that adopts after for improvement includes glucose and plurality of inorganic salt, and alpha-lactose is inductor, added with antibiotic not during fermentation.But every liter of hygroscopic bacterium 50g, the wet bacterium of every gram obtains the thick enzyme of 200U.Should guarantee in the fermenting process that the glucose in the substratum exhausts fully before adding inductor.
The separation and purification of embodiment 4, restructuring pig-human urate oxidase albumen
Preferred purifying process flow process is as follows:
1. from thalline, extract thick enzyme
Centrifugal collection thalline behind the taking-up nutrient solution, with pH 6.8,0.02M phosphoric acid buffer flushing thalline.Add the pH 6.8 of 10ml by the wet bacterium of 1 gram, the 0.1M phosphoric acid buffer is resuspended.The ultrasonic cell-break device is processed broken bacterium.Place high speed freezing centrifuge, collecting precipitation.The pH 10.53 that adds original volume in the precipitation, the 0.1M carbonic acid buffer, fully behind the dissolution precipitation, centrifugal, separate supernatant and be thick enzyme.
2. thick enzyme is carried out purifying with ion exchange column DEAE Sepharose Fast Flow.
With pH 10.53, the 0.1M carbonic acid buffer is done A liquid, and with adding the pH 10.53 that contains 0.5M sodium-chlor, the 0.1M carbonic acid buffer is done B liquid, carries out gradient elution, Fractional Collections, and the higher eluted protein liquid of purity is reclaimed in the protein electrophoresis analysis, is used for being further purified.
3. carry out purifying with drainage column Phoenyl Sepharose 6 Fast Flow (high.sub)
Contain the pH 10.53 of 1.2M sodium-chlor, the 0.1M carbonic acid buffer is done A liquid, and pure water is done B liquid, gradient elution, Fractional Collections protein peak.It is active to measure the urico-oxidase of collecting liquid, and carries out the protein electrophoresis analysis, reclaims the higher elutriant of purity, is used for being further purified.
4. add in the enzyme liquid that drainage column reclaims with 3M acetic acid, regulate pH to 6.8, put under the 20000rpm collecting precipitation centrifugal 20 minutes.
With above-mentioned throw out with an amount of volume pH 10.53, the 0.1M carbonic acid buffer fully dissolves, to reduce enzyme liquid long-pending as far as possible.Carry out sieve chromatography with Sephacryl S-300HR after filtering.Collection contains the albumen main peak of urico-oxidase.
6. the SDS-PAGE of purifying protein detects, and the results are shown in accompanying drawing A.Master tape is single subunit pig-human urate oxidase as seen from the figure, and molecular weight is about 33KD, and larger protein band is the not dimer of complete depolymerization of pig-human urate oxidase, and molecular weight is about 66KD.The high performance liquid phase detected result of purifying protein is seen accompanying drawing B.
7. use SE-HPLC-multiple angle laser light scattering instrument-differential refractometer, mensuration purifying unmodified pig-human urate oxidase relative molecular weight result is: Molar Mass Moments (g/mol)
Mn：1.313e+05(0.7％)
Mw：1.313e+05(0.7％)
Mz：1.314e+05(1.6％)
Thus the result as can be known, the unmodified pig-human urate oxidase relative molecular weight behind the purifying is: 131.3KD.Because purifying protein SDS-PAGE sex change electrophoresis result shows that the molecular weight of zymoprotein list subunit is about 33KD, infers that thus the pig-human urate oxidase behind the purifying mainly exists with tetramer form.
The preparation of embodiment 5, PEGization restructuring pig-human urate oxidase binding substances
The restructuring pig-human urate oxidase that exists with tetramer form of purifying is made into the solution of 5-10mg/ml with the carbonic acid buffer of 0.1M pH10.53.Calculate and add activated PEG product (mPEG-SC, the molecular weight: 10K) that Beijing Kaizheng Biotech Engineering Development Co., Ltd. produces.Method of calculation: mPEG-SC-10K (mg)=(13.5 * purification of Recombinant pig-human urate oxidase mg to be finished)/3.3
By calculated amount weighing m PEG-SC-10K, get first 1/2 of total amount and slowly join in the purifying enzyme liquid, constantly stir enzyme liquid in the adition process, avoid partial concn too high.Under 15-25 ℃ of condition, continue after adding to stir 30 minutes.Get again 1/4 of calculated amount, join in the same way in the enzyme liquid, stir after 30 minutes remaining mPEG-SC-10K joined in the enzyme liquid and stirred 30 minutes.
The purifying of PEGization urico-oxidase, with Sephacryl S-300HR post 0.1M, then pH7.4 phosphoric acid buffer balance with PEGization restructuring pig-human urate oxidase upper prop, uses 0.1M, and pH7.4 phosphoric acid buffer wash-out is collected the protein peak that contains enzyme.
Subunit Lys modifies the measuring method of number:
Use SE-HPLC-multiple angle laser light scattering instrument-differential refractometer, measure the relative molecular weight of the pig-human urate oxidase after unmodified pig-human urate oxidase and PEG modify.Then calculate Lys-ε that how many PEG molecules and tetramer pig-human urate oxidase surface arrangement are arranged-NH2 and form covalent linkage.And then calculate each subunit enzyme molecular surface and be combined with how many PEG molecules.After modifying, several different modifying methods the results are shown in Table 1.
Table 1.SE-HPLC multiple angle laser light scattering instrument-differential refractometer detects modification effect
Pig-human urate oxidase after the various modification mode PEGization still exists with tetramer form.
PEGization restructuring pig-human urate oxidase is all solvable in the carbonic acid buffer of two kinds of different pH, and is also solvable in the phosphoric acid buffer of pH7.4.This purification and clinical application for zymin all is very helpful.Dissolve in restructuring pig-human urate oxidase chimeric protein and the carbonic acid buffer of pig urico-oxidase more than pH10.2.The dissolution characteristics of the restructuring pig-human urate oxidase of PEGization sees Table 2.
The dissolution characteristics of table 2.PEGization restructuring pig-human urate oxidase
The restructuring of PEGization described in following examples pig-human urate oxidase exists with tetramer form.
The activity ratio of embodiment 6, PEGization restructuring pig-human urate oxidase and PEGization restructuring pig-human urate oxidase not
PEGization restructuring pig-human urate oxidase and PEGization restructuring pig-human urate oxidase are not carried out respectively vitality test and the calculating of remaining vigor.Concrete grammar is as follows:
The definition of enzyme assay and unit of activity at 25 ℃, is got the 120 μ mol/L uric acid solution that dissolve in 3mL pH 8.4 borate buffers with reference to the method [3] of Koyama etc., in the 1cm quartz cuvette, adds an amount of urico-oxidase solution to be measured.The abundant rearmounted UV-1601PC ultraviolet spectrophotometer of mixing, accurate response 5min does blank utmost point zero point with borate buffer, in the reduction of 293nm place mensuration light absorption value.Unit of activity is defined as, and the interior catalysis 1 μ mol uric acid of 1min becomes the needed enzyme amount of wallantoin under this condition.Its calculation formula is:
In the formula: the every mL urico-oxidase of U=unit of activity number; Optical density(OD) drop-out value under the Δ A=per minute 293nm wavelength; Vt=reaction solution cumulative volume (mL); The df=extension rate; 12.2 be the micromole optical extinction coefficient of uric acid under the 293nm wavelength; Ve=enzyme liquid amasss (mL).
The remaining vitality test of modifying enzyme
Survey active method by aforementioned enzyme and measure respectively enzyme activity after PEG modifies and the enzyme activity of unmodified, then calculate its remaining vigor by following formula.
After using the mPEG-SC 10KD of several different ratioss that the purification of Recombinant pig-human urate oxidase is modified, measure modify before and after enzymic activity the results are shown in Table 3.
The restructuring pig-human urate oxidase is active before and after the table 3. different modifying method
Annotate: PEG/ urico-oxidase (mol ratio) is 13.5 o'clock, and before enzyme activity surpassed modification after modifying, its reason had two.One, the PEG of this concentration fail to destroy the tetrameric space structure of pig-human urate oxidase, and its activity is uninfluenced; Its two, increased the solvability of enzyme after the PEGization.
1.PEG change the experiment of restructuring pig-human urate oxidase cavy supersensitivity
1. hypersensitive test
1.1 test method
Get 18 of body weight 250-300g healthy guinea pigs, be divided at random 3 groups (13.5 * 10K PEG-PHUO group, 15 * 10K PEG-PHUO group, 16.5 * 10K PEG-PHUO groups) 6 every group.Abdominal injection need testing solution (0.5mg/ml) 0.5mL/ only carries out sensitization, totally 3 times next day of respectively.The 14th, 21 intravenous injection need testing solution (0.5mg/ml) 1mL/ only excite after the last injection, after intravenous injection at once to 30 minutes, the longest observation 3 hours appears and extinction time by describing reaction symptom, the symptom that symptom examines every cavy among 2010 editions Chinese Pharmacopoeia appendix XII G.And whether produce anaphylaxis by the evaluation of 2010 editions Chinese Pharmacopoeia appendix XII G Plays.
1.2 test-results
13.5 behind * 10K PEG-PHUO group, the 15 * 10K PEG-PHUO group intravenously administrable in 30 minutes all cavys anaphylaxis does not all appear, after the administration of 16.5 * 10K PEG-PHUO group in 1 minute 4 cavys nose, 1 cavy occur grabbing continuously cough 3 and retch and four limbs feel like jelly continuously.The results are shown in Table 4.
Table 4 injection PEGization Recombinant Swine urico-oxidase injection liquid Hypersensitive tests result (n=6)
The result shows: under above-mentioned test conditions, systemic anaphylaxis does not appear in injection PEGization Recombinant Swine urico-oxidase 13.5 * 10K PEG-PHUO group, 15 * 10K PEG-PHUO group, 16.5 anaphylaxis appears in * 10K PEG-PHUO, the anaphylaxis incidence is 66.7%.3 experimental group are all used the zymoprotein of same dosage, but have used the PEG of different amounts.13.5 systemic anaphylaxis does not appear in * 10K PEG-PHUO group, 15 * 10K PEG-PHUO group, illustrates that the albumen itself after modifying does not cause obvious anaphylaxis.And anaphylaxis appears in 16.5 * 10K PEG-PHUO, and deduction is because the anaphylaxis that a large amount of modifications causes with PEG.
2.PEG change restructuring pig-human urate oxidase treatment chicken serum antibody ELISA test experience
2.1 chicken anti-restructuring pig-human urate oxidase TPPA and determination methods as a result
ELISA detects the anti-pig-human urate oxidase antibody of chicken serum
40 of experimental chickens are divided into 4 groups, 10 every group.Be respectively 13.5 * 10K PEG-pig-human urate oxidase treatment group, 15 * 10K PEG-pig-human urate oxidase treatment group, unmodified pig-human urate oxidase treatment group and blank group.
Get the carbonic acid soln (0.1M, pH 10.2) of purification of Recombinant pig-human urate oxidase, protein content is 1.2mg/ml, adds 100ul in every micropore, puts 4 ℃ of refrigerator overnight.Next day, wash plate 3 times with PBS-T.Every hole adds the carbonic acid buffer that 200 microlitres contain the pH 9.6 of 5% skim-milk, and sealing is spent the night.With PBS-T wash 3 time after empty the doing next day, and it is dry fixing to put 37 ℃ of 1h.With the rearmounted 4 ℃ of Refrigerator stores of aluminium-foil paper sealing.Every hole adds the chicken serum of 100 microlitres after PBS dilution (1: 400) during mensuration, and does contrast with PBS.37 ℃ of temperature were bathed 30 minutes.Wash 3 times with PBS-T after empty the doing.Every hole adds the anti-chicken HRP of rabbit ELIAS secondary antibody 100 microlitres of dilution in 1: 500, and 37 ℃ of temperature were bathed 30 minutes.Wash 3 times with PBS-T after empty the doing.Every hole adds each 1 of chromogenic substrate A and B, puts the dark place and adds 1 of stop buffer after 10 minutes.Read absorbance value with microplate reader at 450nm wavelength place.Each sample is done 3 parts, results averaged.Judging criterion represents with the ratio (P/N) of serum absorbance to be checked and blank serum absorbance: P/N 〉=2.1 are positive, and P/N＜1.5 are negative, and 1.5≤P/N≤2.1 are suspicious.
2.2 the ELISA detected result of the anti-pig-human urate oxidase antibody of chicken sees Table 5.
The ELISA detected result (n=10) of the anti-pig-human urate oxidase antibody of table 5. chicken
The result shows: the naked enzyme treatment group of pig-human urate oxidase rises since the 14th day P/N value, and the 21st day P/N value surpasses can coagulate positive standard, surpasses the standard of antibody positive, further raises in the 35th day in the 28th day.And 13.5 * 10K PEG-PHUO group and 15.0 * 10K PEG-PHUO group P/N value all do not reach suspicious standard.
Test method
Get mouse, male and female half and half are carried out first trial test, and according to the trial test result, official test is carried out in grouping.Each group is all by the 50ml/kg volume, the different concns intravenously administrable, and injection speed 0.1ml/ second, observation animal immediate reaction and Continuous Observation are 7 after the administration.Dead number of mice respectively organized in record, calculates LD with the Bliss method
50Do not measure LD
50Then carry out the maximum dosage-feeding test.
Test-results
16.5 * 10K PEG-PHUC trial test with maximum administration concentration, maximum administration volume (2.2mg/ml, 50ml/kg) administration after 5 mouse all dead, official test divides 4 groups to carry out LD
50Test, the quickening of official test administration rear section animal breath, tic, the movable minimizing, 6-10 death in second after the dead animal administration, surviving animals recovery in 3-4 minute after administration is normal; Dead number of mice respectively organized in record, and the Bliss method calculates: mouse vein administration 16.5 * 10K PEG-PHUC LD
50Be 69.8mg/kg, 95% the credible 66.6～73.1mg/kg that is limited to.The results are shown in Table 6.
Table 6 16.5 * 10K PEG-PHUO mouse vein administration medium lethal dose
15 * 10K PEG-PHUC trial test with maximum administration concentration, maximum administration volume (2.2mg/ml, 50ml/kg) administration after 5 mouse all dead, official test divides 4 groups to carry out LD
50Test, the animal breath quickening of official test administration rear section, tic, urinary incontinence, the movable minimizing, 4-10 is dead in second after the dead animal administration, and surviving animals recovery in 1-3 minute after administration is normal; Dead number of mice respectively organized in record, and the Bliss method calculates: mouse vein administration 15 * 10K PEG-PHUO LD
50Be 96.7mg/kg, 95% the credible 92.4～101.4mg/kg that is limited to.The results are shown in Table 7.
Table 7.15 * 10K PEG-PHUO mouse vein administration medium lethal dose
13.5 * 10K PEG-PHUC trial test with maximum administration concentration, maximum administration volume (2.2mg/ml, 50ml/kg) administration after 5 mouse all survive, react without overt toxicity.The maximum dosage-feeding test is carried out in official test, and 20 animals are got in official test, and administration rear section animal activity reduces, and recovers normal in 1-2 minute, without other toxic reactions, without animal dead.Its maximum dosage-feeding is 111mg/kg.
The injection preparation of embodiment 9, PEGization restructuring pig-human urate oxidase binding substances
Purifying PEGization restructuring pig-human urate oxidase is mixed with the 1mg/ml enzyme solution with pH7.4 phosphoric acid buffer liquid.Divide after the Sterile Filtration and be filled in the cillin bottle every bottle of 1ml, vacuum seal.2-8 ℃ of preservation.
The lyophilized injectable powder of embodiment 10, PEGization restructuring pig-human urate oxidase binding substances
Purifying PEGization restructuring pig-human urate oxidase is mixed with the 1mg/ml enzyme solution with pH7.4 phosphoric acid buffer liquid, to final concentration 3%, adds glycine to final concentration 2% with N.F,USP MANNITOL.Divide after the Sterile Filtration and be filled in the cillin bottle every bottle of 1ml.Lyophilize, 2-8 ℃ of preservation.
Embodiment 11 PEGization restructuring pig-human urate oxidases reduce the effect of laboratory animal serum uric acid level
The serum uric acid measuring method
Giving birth to the blood uric acid detection kit (enzymic colorimetric) of north control Bioisystech Co., Ltd in the employing measures.Measuring method is: get 5ml 0.1mol/L phosphoric acid buffer (pH7.8), be added to contain urico-oxidase (〉=600U/L), peroxidase (〉=1800U/L), in the reagent bottle of the amino antipyrine (0.3mmol/L) of 4-and TOOS (1.4mmol/L), shake gently to fully dissolving, be working fluid.Then according to the form below operation:
Table 8. serum uric acid is measured the liquid feeding operation
Mix respectively, at 37 ℃ of insulation 10min, detect uric acid concentration in the blood sample with automatic biochemistry analyzer, under 550nm, read respectively A with the blank tube zeroing
StandardAnd A
Sample
Calculate:
Test effective acting time of the lyophilized injectable powder of PEGization restructuring pig-human urate oxidase
24 adult U.S. Wang Ge, body weight is between 500-580 gram/only.Be divided at random 3 groups, 8 every group, be respectively PEG modifying enzyme experimental group (testing 1 group), naked enzyme experimental group (testing 2 groups) and blank group.Test 1 group by 1mg (6U)/kg (body weight) intravenous injection 15 * 10K PEG-PHUO.Before the experiment zymin added 10% glucose injection to 5 milliliter.Test 2 groups by the naked enzyme of restructuring pig-human urate oxidase behind 1mg (6U)/kg (body weight) intravenous injection purifying, add 10% glucose injection to 5 milliliter before the experiment.5 milliliters of blank group intravenous injection 10% glucose injections.Taked respectively to measure serum uric acid behind the venous blood separation of serum in rear 1,6,24,72,120,168,216 hour with injection before the per injection.Interval injection in 14 days is once injected three times altogether.The Plasma Uric Acid assay the results are shown in Table 9.
Plasma Uric Acid content detection result after for the first time injection of table 9.
Annotate: * represents that Plasma Uric Acid content compared significant difference with control group.
By table as seen, modifying enzyme injection group 1 hour Plasma Uric Acid content after administration significantly reduces, and keeps significantly to be lower than the control group uric acid level 7 days.The naked enzyme injection of purifying group was injected rear 1 hour, and Uric Acid Content significantly was lower than control group in 6 hours, and bounce-back in 24 hours surpasses the control group level.Return to later on the control group level.
Plasma Uric Acid content detection result after for the second time injection of table 10.
Annotate: * represents that Plasma Uric Acid content compared significant difference with control group.
For the second time after the injection, experiment dove Uric Acid Content significantly is lower than control group, can keep 7 days, compares not very big difference with for the first time injection, and the 7th day Uric Acid Content rebound significantly just, but still be lower than control group reached level before the injection in 9 days.The naked enzyme injection of purifying group only significantly reduced Uric Acid Content in 1 hour in administration, just substantially returned to the level suitable with control group in 6 hours.
Plasma Uric Acid content detection result after table 11. is injected for the third time
Annotate: * represents that Plasma Uric Acid content compared significant difference with control group.
After the injection, modifying enzyme group Uric Acid Content can be kept within the 5 day time of injection and be lower than the control group level, returns to normal level after 7 days for the third time.
From whole experimental result, the pig-human urate oxidase after the modification obviously prolongs the effective acting time in blood, surpasses not modified purifying enzyme.During interval 2 all repeat administrations, shorten effective acting time to some extent.Inject for the third time and still can reach 5 days effective acting time.Not modified urico-oxidase experimental group only can kept 6 hours low Uric Acid Content after the injection for the first time.And the Uric Acid Content before the rising of rear 24 hours Uric Acid Content bounce-back property of injection surpasses injection.After the injection, only can keep 1 hour low Uric Acid Content for the second time.For the third time not significantly effect after the injection.
Reference:
[1]Friedman TB，Polanco GE，Appold JC，Mayle JE：On the loss of uricolytic activity during primate evolution-I.Silencing of urate oxidase in a hominoid ancestor.Comp Biochem Physiol 81B(3)：653-659，1985.
[2]Amy C，Cannella，Ted R，Mikuls.Understanding treatments for gout.Am J Manag Care.2005；11：S451-S458.
[3]Koyama Y，Ichikawa T，Nakano E.Cloning，sequence analysis and expression in Escherichia coli of the gene encoding the Candidaut ilisurate oxidase(uricase).Biochem，1996，120：969-973.
Claims (12)
1. restructuring pig-human urate oxidase fusion protein, the aminoacid sequence that it is characterized in that this fusion rotein is shown in the sequence 2 in the sequence table.
2. restructuring pig-human urate oxidase fusion protein according to claim 1, the covalently bound formation binding substances of this fusion rotein and polyoxyethylene glycol (PEG), that is: PEGization restructuring pig-human urate oxidase.
3. restructuring pig-human urate oxidase fusion protein according to claim 1, it is characterized in that the reaction of described restructuring pig-human urate oxidase fusion protein and mono methoxy polyethylene glycol formic acid succinimide ester, the PEG of the upper activation of mPEG is connected by covalent linkage with part free amine group in the restructuring pig-human urate oxidase fusion protein molecule, forms PEGization restructuring pig-human urate oxidase.
4. restructuring pig-human urate oxidase fusion protein according to claim 3 is characterized in that covalently bound 8-12 PEG chain on each restructuring pig-human urate oxidase fusion protein subunit, and the molecular-weight average of PEG is 2-50KD, and PEG is linear or branch shape.
5. restructuring pig-human urate oxidase fusion protein according to claim 4 is characterized in that covalently bound 9-11 PEG chain on each restructuring pig-human urate oxidase subunit, and the molecular-weight average of PEG is 5-20KD, and PEG is linear or branch shape.
6. restructuring pig-human urate oxidase fusion protein according to claim 5 is characterized in that covalently bound 9-10 PEG chain on each restructuring pig-human urate oxidase subunit, and the molecular-weight average of PEG is 10KD, and PEG is linear or branch shape.
7. a nucleic acid molecule is characterized in that, its nucleotide sequence is shown in sequence in the sequence table 1.
8. plasmid vector contains nucleic acid molecule claimed in claim 7.
9. host cell contains plasmid vector claimed in claim 8, is e. coli bl21, is preserved in Chinese Typical Representative culture collection center, and deposit number is CCTCC M2011161.
10. a composition that reduces humans and animals blood uric acid content is characterized in that comprising PEGization restructuring pig-human urate oxidase fusion protein claimed in claim 1 and pharmaceutically acceptable carrier.
11. composition claimed in claim 10 is characterized in that said composition exists with injection liquid or freeze-dried powder form.
12. the described restructuring pig-human urate oxidase fusion protein of claim 1-11 is for the preparation of the application in the medicine that reduces humans and animals blood uric acid content.
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| CN105412942B (en) * | 2015-12-23 | 2019-02-26 | 沈阳三生制药有限责任公司 | The recombination candida utili urate oxidase freeze dried injection of Pegylation |
| CN108103079B (en) * | 2017-06-20 | 2021-07-13 | 北京锦篮基因科技有限公司 | Gene therapy medicine for hyperuricemia |
| CN113144174B (en) * | 2020-01-22 | 2023-07-04 | 杭州远大生物制药有限公司 | Medicine for treating hyperuricemia related diseases |
| CN111500550A (en) * | 2020-04-02 | 2020-08-07 | 北京翼方生物科技有限责任公司 | Filter medium capable of degrading human blood uric acid and preparation method thereof |
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| WO2003011211A2 (en) * | 2001-08-02 | 2003-02-13 | Phoenix Pharmacologics, Inc. | Peg-modified uricase |
| US7056713B1 (en) * | 1998-08-06 | 2006-06-06 | Duke University | Urate oxidase |
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| CN102051348A (en) * | 2009-10-27 | 2011-05-11 | 重庆富进生物医药有限公司 | Humanized recombinant uricase and mutant thereof |
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| CN1322141A (en) * | 1998-08-06 | 2001-11-14 | 山景药品公司 | PEG-urate oxidase conjugates and use thereof |
| US7056713B1 (en) * | 1998-08-06 | 2006-06-06 | Duke University | Urate oxidase |
| WO2003011211A2 (en) * | 2001-08-02 | 2003-02-13 | Phoenix Pharmacologics, Inc. | Peg-modified uricase |
| CN101198693A (en) * | 2005-04-11 | 2008-06-11 | 萨文特医药公司 | Variant forms of urate oxidase and use thereof |
| CN102051348A (en) * | 2009-10-27 | 2011-05-11 | 重庆富进生物医药有限公司 | Humanized recombinant uricase and mutant thereof |
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